莱菔硫烷抑制LPS诱导的小鼠星形胶质细胞增殖

目的 探讨莱菔硫烷(sulforaphane,SFN)对脂多糖(LPS)诱导的星形胶质细胞(astrocyte,AST)增殖的影响及其机制.方法 体外培养小鼠原代星形胶质细胞,给予0.1 μg/mL LPS刺激星形胶质细胞活化增殖;同时分别给予不同浓度的莱菔硫烷(10、15、20 μmol/L)处理星形胶质细胞24 h后,CCK8法检测细胞增殖情况;选取20 μmoL/L作为干预剂量行GFAP、Ki67双标免疫荧光染色及流式细胞仪细胞周期检测,进一步验证莱菔硫烷对LPS诱导星形胶质增殖的作用;Western blot检测细胞周期相关蛋白P27kip1、CyclinD1、PCNA的表达情况.结果 CCK8法检测显示0.1μg/mL LPS能显著促进星形胶质细胞增殖(P＜0.01),而加入20μmol/L莱菔硫烷能显著抑制LPS诱导的星形胶质细胞增殖(P＜0.01),并能减少LPS刺激下的Ki67阳性细胞(P＜0.05);细胞周期分析提示20 μmol/L莱菔硫烷可减少LPS诱导下的星形胶质细胞S期比例,增加G1期细胞比例(P＜0.05);Western blot提示20 μmol/L莱菔硫烷能下调LPS刺激下PCNA、CyclinD1的表达,上调P27kip1表达(P＜0.05).结论 莱菔硫烷可以通过调控星形胶质细胞细胞周期,抑制体外LPS诱导的星形胶质细胞增殖.

Sulforaphane inhibits astrocyte proliferation by induced LPS in mice

Objective To determine the effect of sulforaphane (SFN) on the proliferation of astrocytes induced by lipopolysaccharide (LPS).Methods Primary astrocytes were isolated from the mouse cortex and then cultured in vitro.After the cells were stimulated with 0.1 μg/mL LPS in the absence or presence of different concentrations of SFN (10,15 and 20 μmol/L) for 24 h,the cell proliferation was tested with CCK8 assay.Immunofluorescent double staining and cell cycle analysis were performed respectively in the cells treated with 20 μmol/L SFN to further verify the effect of SFN on the proliferation of astrocytes after LPS stimulation.The expression levels of cell cycle related proteins,P27kip1,Cyclin D1 and proliferating cell nuclear antigen (PCNA) in each group of cells were determined by Western blotting.Results CCK8 assay indicated that 0.1 μg/mL LPS promoted the proliferation of astrocytes (P ＜0.01).Treatment of 20 μ mol/L SFN inhibited the proliferation significantly (P ＜ 0.01) and reduced the number of Ki67 positive cells under LPS stimulation (P ＜ 0.05).Cell cycle analysis showed that 20 μmol/L SFN treatment attenuated the increment of cells in S phase and the reduction of cells in G1 phase which were induced by LPS stimulation (P ＜0.05).Western blot assay represented that LPS enhanced the expression of PCNA and Cyclin D1,meanwhile,decreased the level of P27kip1 (P ＜ 0.05).Conclusion SFN inhibits the proliferation of astrocytes induced by LPS,which may be through regulation of cell cycle.